Journal of ChineseMedicinalMaterials H 2 O 2. Effects of EGb 761 on the Cell Apoptosis Induced by H 2 O 2 in R IN 2m Beta Cells

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1 424 H 2 O 2 R IN2m,,, (, ) : ( Ginkgo biloba extract, EGb761) (Hydrogen peroxide, H 2 O 2 ) RIN2m : 500 mol/l H 2 O 2 RIN2m 6h ; (Control) (H 2 O 2 ) ( Que 100 mol/l) EGb 761 ( EGb mol/l), EGb 761 ( EGb g/ml) ; MTT, Hoechst 33258,PI Annexin V2PI :, 500 mol/l H 2 O 2 6h, ( P<0101), EGb 761 H 2 O 2 ( P<0101), : RIN2m, EGb 761 H 2 O 2 RIN2m ;RIN2m ; ; : R28515 : A : (2007) Effects of EGb 761 on the Cell Apoptosis Induced by H 2 O 2 in R IN 2m Beta Cells YE Chun2ling, J IN Yong2liang, YE Kai2he, Q IN Liang (Department of Pharmacology, Pharmacy College of J inan University, Guangzhou , China) Abstract O bjevtive: To investigate the effects of Ginkgo biloba extract ( EGb 761) on apop tosis induced by hydrogen peroxide (H 2 O 2 ) in R IN2m beta2cells1 Methods: The apop totic model was made by H 2 O 2 exposed for six hours with a concentration of 500 mol/l1 The cytotoxicitywasmeasured bymtt1 Hoechst fluorescent staining were used to detect the p rotective effect of EGb 761 on the apop tosis of R IN2m beta2cells induced by H 2 O 2 1 AnnexinV2P I double staining of Flow cytometry were used to detect apop2 to sis quan titively1 Results: Compare to control group, after exposed to 500 mol/l H 2 O 2 for 6 hours, the apop tosis rate incereased and cell survival rate were decreased considerably ( P <0101) 1 Pretreated for 10 hourswith EGb 761, the flow cytometry results showed that the apop to sis rate decreased and cell survival rate were increased considerably ( P<0101, compared to H 2 O 2 con tro l group ) 1 Conclu2 sion: EGb 761 can decrease R IN2m beta2cells damage and apop tosis induced by H 2 O 2 1 Key words EGb 761; R IN2m beta2cells; H 2 O 2 ; Apoptosis,, 1 2,,,,, H 2 O 2 RIN2m,, EGb ( Extract Ginkgo biloba, EGb 761)( : ) ;515 mmol/l DM EM Gibco ;, ; 2 Hoechst, ; ( Tryp sin), 2( 4, 52,22 ) 22, 52 (MTT), ( p rop idiuiodide, P I) Sigma,Annexin2V PI ( : ) 112 RIN2m 10% ( 100 U /m l) ( 100 g/ml) 515 mmol/l DM EM, 37 5% CO 2 : 3 :,, Tel: , E2mail: yechunling2005@1631com

2 Journal of ChineseMedicinalMaterials ( FORMA, U1S1A) ; 5084R ( EPPENDORF, Germ any) ;FACS Aria ( BD ) ; 450 (BIO2RAD, USA ) ;22DI2E2D282 (L E ICA, Ge rm any) H 2 O 2 RIN2m 10 5 /m l 96,37,5%CO 2, ( control) ( H 2 O 2 ) ( Que 100 mol/l) EGb 761 ( EGb mol/ L),EGb761 ( EGb mol/l), h, 500 mol/l H 2 O 2 6h, MTT MTT , PB S, 012mlPBS 20 l MTT(5mg/ml), 015 mg/ml 37 4h, 150 l DM SO 37, 570nm/630nm (op tical density, OD),, 100%, = / 100% 213 Hoechst 33258, 50 80% 6,,,600 g/min 5min ; 4% 015 ml, 10 m in ( 4 ) ;,PBS 2, 3 min,,, ; 600 g/m in 5min PB S; 015 mlhoechst min, ; PB S 2, 3min,,,, ; 350 nm, 460 nm 214 ( FCM ) RIN2m : /m l, 0125% 20102% EDTA,3mlPBS, 70%,4 24 h,3 ml PBS 5min, 1% Triton X m in,, 0101% RNA 10 m in,, 01005% PI, 4 30 m in, 400, PI, 488 nm, 630 nm,, Cell Quest, ModFit LT RIN2m : 0125% 20102% EDTA,, /m l, Annexin2VPI, PI 215 ( gx S), t SPSS H 2 O mol/l H 2 O 2, RIN2 m ( P<0101) g/ ml EGb 761 H 2 O 2, H 2 O 2 ( P<0105 P< 0101), 1 Tab 1 Effect of EGb 761 on R IN2m beta2cells in the Presence of 500 M H 2 O 2 ( gx s, n =6) Group s V iability( % ) Control H 2 O mol/l ## EGb g/ml Que +H 2 O mol/l EGb H 2 O 2 10 g/ml g/ml g/ml P<0105, 33 P<0101 vs H 2 O 2 ; ## P<0101 vs Control 312,,,, DNA EGb 761,, EGb g/ml, H 2 O 2 EGb 761 H 2 O 2 1

3 426 F ig111 Protection of EGb 761 Aga in st Apoptosis Induced by H 2 O 2 in R IN2m 2cells( 400 ) A1 H 2 O 2 ; B1 H 2 O g/ml EGb761; C1 H 2 O g/ml EGb761; D1 H 2 O g/m l EGb761; E1 Control; F1 H 2 O 2 + Que Fig12 Protection of EGb 761 Aga inst H 2 O 2 2Induced Apoptosis in RIN2m 2cells( P I) 33 P<0101 vs H 2 O 2 without EGb 761, ## P<0101 vs Control 313 RIN2m PI : PI 2 3 H 2 O 2 H 2 O 2, RIN2m G 0 /G 1 ( sub2g 1 ), 500 mol/l H 2 O 2 6h RIN2m ( % ) ;EGb761,, EGb 761 ( % ) Que ( % ),, ( P<0101) 3, EGb 761,, AnnexinV2PI :Annex2 in V 2PI 4 5 ( PS),AnnexinV PS; PI, DNA, Annexin V2 FITC/PI,H 2 O 2 6h 4, (An2 nexin V 22 2PI + ) (Annexin V 22 2PI 22 ) (Annexin V + 2PI 22 ) (Annexin V + 2PI + ) H 2 O mol/l H 2 O 2, RIN2m 6h ( ) %, ( ) %, ( P<0101) ; 100 g/ml EGb 761 ( ) % Que ( ) % H 2 O 2, ( P<0101) 4,, 5,

4 Journal of ChineseMedicinalMaterials F ig131 Apoptosis in R IN2m 2cells A ssessed by FCM A1 H 2 O 2 ; B1 H 2 O g/ml EGb761; C1 H 2 O g/ml EGb761; D1 H 2 O g/m l EGb761; E1 Control; F1 H 2 O 2 + Que, Annexin V2PI,, PI, Annexin V PI, FCM An2 nexin V2PI,,,, RIN2m H 2 O 2 6 7,PI Annexin V2PI, EGb 761 Fig14 Protection of EGb 761 Aga inst H 2 O 2 2Induced H 2 O 2 RIN2m, Apoptosis in RIN2m 2cells( Annex in V2PI), EGb 761 H 33 P<0101 vs H 2 O 2 without EGB 761, ## 2 O 2 P<0101 vs Control,,,,, ROS,,, 8 H 2 O 2,, H 2 O 2 PC12 RO S 2 9 EGb AnnexinV,AnnexinV, 10 11,EGb,, NO Annexin V,, EGb

5 428 F ig151 Apoptosis in R IN2m, [ 1] VasseurM, Jean T, DeFeudis FV, et al1 Effects of repeat2 ed treatments with an extract of Ginkgo biloba ( EGb 761), bilobalide and ginkgolide B on the electrical activity of pancreatic beta cells of normal or alloxan2diabetic m ice: an exvivo study with intracellular m icroelectrodes1 Gen Phar2 macol, 1994, 25 (1) : [2],,, 1 1,2000,11( 3 ) : [3], 1 1 :, [4] MortenBH, Svend EN, KurtB1 Re exam ination and fur2 ther development of ap recise and rap id dye method form easuring cell growth /c kill1 J Immunol Meth, 1989, 119: [5] O Biren BA, Harmon BV, Cameron DP, et al1 Beta2cell apop tosis is responsible for the dvevlopment of IDDM in the 2cells a ssesssed by FCM multip le low2dose strep tozotocin model1 J Pathol, 1996, 178 (2) : [6] Thomas G, Masanobu I, Coretta vanl, et al1 H 2 O 2 inhib2 its alveolar ep ithelial wound repair in vitro by induction of apop tosis1 AJP2Lung, 2004, 287: [ 7 ] Hao Hong, Guo2Q ing L iu1 Protection against hydrogen per2 oxide2induced cytoxicity in PC12 cells by scutellarin1 Life Sciences, 2004, 74: [8] 1 1 :, [ 9 ] Takum i S, Naoto S, Yasushi E, et al1 Free radical inde2 pendent protection by nerve growth factor and Bcl22 of PC12 cells from hydrogen peroxide triggered apop tosis1 J B io chem, 1996, 120: [10],, 1 1,2003,23(5): [11] 1, 1996, 21 (4) : ( ),!